Glycoproteins exhibit a variety and complexity of carbohydrate units, the composition and arrangement of the carbohydrates being characteristic of different organisms. The oligosaccharide units of the glycoproteins have a number of tasks, e.g. they are important in regulating metabolism, they are involved in transmitting cell-cell interactions, they determine the circulation periods of proteins in circulation, and they are decisive for recognizing epitopes in antigen-antibody reactions.
The glycosylation of glycoproteins starts in the endoplasmatic reticulum (ER), where the oligosaccharides are either bound to asparagine side chains by N-glycosidic bonds or to serine or threonine side chains by O-glycosidic bonds. The N-bound oligosaccharides contain a common core from a penta-saccharide unit which consists of three mannose and two N-acetyl glucose amine residues. To further modify the carbohydrate units, the proteins are transported from the ER to the Golgi complex. The structure of the N-bound oligosaccharide units of glycoproteins is determined by their conformation and by the composition of the glycosyl transferases of the Golgi compartments in which they are processed.
It has been shown that the core pentasaccharide unit in the Golgi complex of some plant and insect cells is substituted by xylose and α1,3-bound fucose (P. Lerouge et al., 1998, Plant Mol. Biol. 38, 31-48; Rayon et al., 1998, L. Exp. Bot. 49, 1463-1472). The heptasaccharide “MMXF3” forming constitutes the main oligosaccharide type in plants (Kurosaka et al., 1991, J. Biol. Chem., 266, 4168-4172). Thus, e.g., the horseradish peroxidase, carrot β-fructosidase and Erythrina cristagalli comprise lectin as well as the honeybee venom phospholipase A2 or the neuronal membrane glycoproteins from insect embryos α1,3-fucose residues which are bound to the glycan core. These structures are also termed complex N-glycans or mannose-deficient or truncated N-glycans, respectively. The α-mannosyl residues may be further replaced by GlcNAc, to which galactose and fucose are bound so that a structure is prepared which corresponds to the human Lewis a-epitope (Melo et al., 1997, FEBS Lett 415, 186-191; Fitchette-Laine et al., 1997, Plant J. 12, 1411-1417).
Neither xylose nor the α1,3-bound fucose exist in mammalian glycoproteins. It has been found that the core-α1,3-fucose plays an important role in the epitope recognition of antibodies which are directed against plant and insect N-bound oligosaccharides (I. B. H. Wilson et al., Glycobiology Vol. 8, No. 7, pp. 651-661, 1998), and thereby trigger immune reactions in human or animal bodies against these oligosaccharides. The α1,3-fucose residue furthermore seems to be one of the main causes for the widespread allergic cross reactivity between various plant and insect allergens (Tretter et al., Int. Arch. Allergy Immunol. 1993; 102:259-266) and is also termed “cross-reactive carbohydrate determinant” (CCD). In a study of epitopes of tomatoes and grass pollen, also α1,3-bound fucose residues were found as a common determinant, which seems to be the reason why tomato and grass pollen allergies frequently occur together in patients (Petersen et al., 1996, J. Allergy Clin. Immunol., Vol. 98, 4; 805-814). Due to the frequent occurrence of immunological cross reactions, the CCDs moreover mask allergy diagnoses.
The immunological reactions triggered in the human body by plant proteins are the main problem in the medicinal use of recombinant human proteins produced in plants. To circumvent this problem, α1,3-core-fucosylation would have to be prevented. In a study it could be demonstrated that oligosaccharides comprising an L-galactose instead of an L-fucose (6-deoxy-L-galactose) nevertheless are biologically fully active (E. Zablackis et al., 1996, Science, Vol. 272). According to another study, a mutant of the plant Arabidopsis thaliana was isolated in which the N-acetyl-glucosaminyl transferase I, the first enzyme in the biosynthesis of complex glycans, is missing. The biosynthesis of the complex glycoproteins in this mutant thus is disturbed. Nevertheless, these mutant plants are capable of developing normally under certain conditions (A. Schaewen et al, 1993, Plant Physiol. 102; 1109-1118).
To purposefully block the binding of the core-α1,3-fucose in an oligosaccharide without also interfering in other glycosylation steps, merely that enzyme would have to be inactivated which is directly responsible for this specific glycosylation, i.e. the core-α1,3-fucosyl transferase. It has been isolated and characterized for the first time from mung beans, and it has been found that the activity of this enzyme depends on the presence of non-reducing GlcNAc ends (Staudacher et al., 1995, Glycoconjugate J. 12, 780-786). This transferase which only occurs in plants and insect, yet not in human beings or in other vertebrates, would have to be inactivated on purpose or suppressed so that human proteins which are produced in plants or in plant cells or also in insects or in insect cells, respectively, do no longer comprise this immune-reaction-triggering epitope, as has been the case so far.
The publication by John M. Burke “Clearing the way for ribozymes” (Nature Biotechnology 15:414-415; 1997) relates to the general mode of function of ribozymes.
The publication by Pooga et al., “Cell penetrating PNA constructs regulate galanin receptor levels and modify pain trans-mission in vivo” (Nature Biotechnology 16:857-861; 1998) relates to PNA molecules in general and specifically to a PNA molecule that is complementary to human galanin receptor type 1 mRNA.
U.S. Pat. No. 5,272,066 A relates to a method of changing eukaryotic and prokaryotic proteins to prolongue their circulation in vivo. In this instance, the bound oligosaccharides are changed with the help of various enzymes, among them also GlcNAc-α1→3(4)-fucosyl transferase.
EP 0 643 132 A1 relates to the cloning of an α1,3-fucosyl transferase isolated from human cells (THP-1). The carbohydrate chains described in this publication correspond to human sialyl Lewis x- and sialyl Lewis a-oligosaccharides. The specificity of the enzyme from human cells is quite different than that of fucosyltransferase from plant cells.